Fluorescence resonance energy transfer-based method for detection of DNA binding activities on nuclear factor κB

Abstract

The DNA binding protein nuclear factor κB (NF-κB) and the cellular signaling pathways in which it participates are the central coordinators of many biological processes, including innate and adaptive immune responses, oxidative stress response, and aging. NF-κB also plays a key role in diseases, for example, cancer. A simple, convenient, and high-throughput detection of NF-κB activation is therefore important for systematically studying signaling pathways and for screening therapeutic drug targets. We describe a method based on fluorescence resonance energy transfer (FRET) to directly measure the amount of activated NF-κB. More specifically, a double-stranded DNA (dsDNA) probe was designed to contain a pair of FRET fluorophores at the same end of the probe and an endonuclease binding site within the NF-κB consensus sequence. The activated NF-κB was detected by FRET following the restriction enzyme digestion. Using three different analyte materials—(i) purified recombinant NF-κBp50, (ii) nuclear extracts, and (iii) whole cell lysates—we demonstrated that this assay is as sensitive as the traditional, widely used electrophoretic mobility shift assay (EMSA), but much less labor-intensive for measuring NF-κB DNA binding activities. In addition, this FRET-based assay can be easily adapted for high-throughput screening of NF-κB activation.